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  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/6858—Allele-specific amplification
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6816—Hybridisation assays characterised by the detection means
  • C12Q1/6825—Nucleic acid detection involving sensors
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6869—Methods for sequencing

Definitions

• the present invention relates to a method for increasing the detectable ionic signal from a chemical reaction, particularly though not exclusively methods for DNA sequencing and SNP identification.
• Sequencing of nucleic acids can be accomplished by synthesising all or part of a nascent nucleic acid strand, in a polymerisation reaction between the unknown (target) polynucleic acid and the free nucleotides dATP, dCTP, DGTP, DTTP added one at a time. Identification is traditionally done by then monitoring the main product of such polymerisation reaction, namely the newly synthesised polynucleic acid by gel electrophoresis, and direct or indirect optical quantification.
• ISFET ION Sensitive Field Effect Transistor
• the chemical reaction is preferably the addition or removal of a particular nucleic acid/nucleotide/nucleoside (the terms being used interchangeably herein).
• a method of identifying an unknown nucleic acid comprising:
• the method comprises determining if there is a significant change in the output signal and correlating only such significant changes.
• the known nucleotide is one of dATP, dGTP, dTTP, or dCTP and the known nucleic acid is a primer.
• a method according to any preceding claim wherein the steps of the method are repeated to sequence the unknown nucleic acid, each repeat using one of dATP, dGTP, dTTP, or dCTP as the known nucleotide.
• the known nucleic acid is an allele-specific primer.
• the polymerisation reaction is an allele-specific amplification reaction
• one of the by-products is pyrophosphate and the one or more enzymes comprise a pyrophosphatase.
• the pyrophosphate may preferably be added to the reaction chamber before the polymerisation reaction starts, such that the polymerisation reaction and deconstruction reaction are substantially concurrent.
• one of the by-product is DNA and one or more of the enzymes comprises an enzyme to deconstruct DNA.
• the one or more enzymes comprise one or more of an exonuclease, preferably selected from the group consisting of: T7 Exonuclease, RecJf, Exonuclease I, Exonuclease T, and BAL-31 Exonuclease, Exonucelase III or Lambda Exonuclease.
• an exonuclease preferably selected from the group consisting of: T7 Exonuclease, RecJf, Exonuclease I, Exonuclease T, and BAL-31 Exonuclease, Exonucelase III or Lambda Exonuclease.
• the one or more enzymes may preferably be added to the reaction chamber after the polymerisation reaction starts such that the deconstruction reaction occurs after the polymerisation reaction.
• the electrical output signal may preferably be measured differentially between the ISFET and another ISFET or MOSFET.
• Determining if there is a significant change in the output signal may preferably comprise determining a magnitude of an electrical output signal change, or more preferably comparing the change in the electrical output signal to a threshold signal change value.
• determining if there is a significant change in the output signal may preferably comprise evaluating the change in the output signal from the polymerisation reaction separately from the change in the output signal from the deconstruction reaction.
• Determining if there is a significant change in the output signal may also comprise comparing the output signal change to an output signal change where the polymerisation reaction does not occur because the one or more nucleotides of the unknown nucleic acid are not complementary to the known nucleotide and/or known nucleic acid.
• An advantage of the present invention is therefore an increase in the signal strength by detecting protons from both the polymerisation and deconstruction reactions. It is particularly preferred that the enzyme hydrolyse pyrophosphate, but not triphosphate acid. Thus, in their presence, a greater pH change can be detected (on top of that from the hydrolysis of dNTP).
• An allele-specific primer for an SNP may be extended by addition of Nucleoside Triphosphates (dNTPs or ddNTPs) during DNA polymerisation.
• the extended/polymerised strand is optionally amplified and then an exonuclease can be added to remove the newly-added nucleotides (or their amplified equivalents) one by one.
• an exonuclease can be added to remove the newly-added nucleotides (or their amplified equivalents) one by one.
• the H+ ions released on both NTP addition and subsequent removal are detected by the ISFET(s).
• Exonuclease activity on non-target DNA can preferably be ameliorated by capping or modification.
• exonuclease activity on non-target DNA is largely irrelevant as it is swamped by the amplified target - this applies to the detection of SNPs in particular, so amplification is particularly preferred for this aspect.
• amplification is probably not always necessary, but if exonuclease is to be used in such sequencing, then the primer that is extended to initiate the sequencing may preferably be capped or modified to prevent exonuclease activity against it.
• the present invention thus provides a method of identifying a nucleic acid. This involves determining its identity, for instance that of its base: i.e. is it C, G, T A or U. This may preferably be part f a method of sequencing a strand of DNA or other genetic material. It may be preferable to use only a PPase to assess levels of PPi produced on addition by polymerisation of a nucleoside to a nascent strand: this may be repeated many times to sequence a long stretch or occur only once. In the latter ascpet, the invention thus also applies to a method of determining the identity of a single nucleotide, in either a larger sequencing effort or in the detection of an SNP.
• nucleoside triphosphate say Cytosine (C).
• C Cytosine
• SNP detection In that case, or where it is desired to detect a the identity of broader stretch of nucleic acids in the template, then it is also particularly preferred expose the template or target strand to primers.
• suitable nucleoside triphosphates i.e. all or at least A, T (or U), C and G
• exonuclease activity is most preferably used in respect of SNP's, particularly to confirm the addition of a nucleoside at the end of a primer, where said end is designed to overly exactly the position of the SNP such that the first or next nucleoside added to the 3' end of the primer will be that complementary to the SNP. Capping and modification of the primer are also helpful here to prevent the primer being acted on.
• FIG. 1 is an exemplary flow chart of a method according to an embodiment of the invention employing a pyrophosphatase (PPase) enzyme;
• PPase pyrophosphatase
• Figure 2 is a graph of change in pH over time for various reactions in both the presence and absence of pyrophosphate (PPi) and pyrophosphatase (PPase);
• Figure 3 is a graph of change in pH over time for various reactions according to an embodiment of the invention employing an exonuclease, in this instance Lambda Exonuclease; and Figure 4 is a graph of change in pH over time for various reactions according to an embodiment of the invention employing an exonuclease, in this instance Exonuclease III.
• Described herein is a detection method which monitors deconstruction reactions associated with a newly-synthesised nucleic acid.
• the substrate of this deconstruction reaction arises from nucleic acid polymerisation (the primary reaction) which itself release hydrogen ions as nucleosides are added to (i.e. polymerised to form) an extending chain.
• the deconstruction reaction is referred to as the 'secondary reaction'.
• This detection of further hydrogen ions released from the secondary reaction can be applied alone, or in addition to the detection of the hydrogen ions released from the primary reaction.
• the creation of a secondary reaction which involves catalysis (principally hydrolysis) of the by-product of primary reaction, initiates an additional secondary signal to the primary signal. Any secondary reaction that is detected by the same detector as the primary reaction can be viewed as a signal enhancement in situ. Hydrogen ions released from secondary reaction can also be detected by a separate detector.
• the aim is signal enhancement, thus enabling increased accuracy over the background. This is achieved by in fact harnessing the background.
• Hydrogen ions released from the secondary reaction are preferably detected by the same (i.e. a single) ISFET sensor as the primary reaction, but it could also be a further sensor, for example if two sensors per reaction chamber are used or if the primary and secondary reactions occur at different times or locations.
• a deconstruction reaction is for instance a reaction, preferably catalysed by the present enzymes, that produces one or more break-down products from a substrate.
• the preferred enzymes are pyrophosphates or exonucleases, both of which are hydrolases which catalyze the hydrolysis of phosphate bonds, thus deconstructing the phosphate bond to ultimate yield more Hydrogen ions.
• the deconstruction enzymes are those capable of hydrolysing phosphate/phosphodiester bonds, especially in the context of nucleic acids (i.e. nucleic acid phosphodiester bonds).
• the (primary) chemical reaction is the synthesis of a nascent polynucleotide strand (being two or more nucleotides) of which RNA and DNA are particularly preferred. It is preferred that the fluctuations of ionic charge indicate the insertion of di- deoxynucleotide triphosphates (ddNTP) and deoxynucleotide triphosphates (dNTP) onto the nascent polynucleotide strand.
• the nascent polynucleotide strand may be an entirely new polynucleotide strand or, alternatively, the newly added sections of an annealed strand such as a primer, added during the preferred sequencing reaction.
• the nascent polynucleotide strand is the complement of a template strand.
• DNA sequencing using an embodiment of the invention is performed as follows, with reference to DNA as the preferred polynucleotide.
• a quantity of DNA of interest is amplified using either a polymerase chain reaction or cloning or other amplification technique, and the region of interest is primed, e.g. using an mRNA primer.
• DNA polymerase catalyses DNA synthesis (polymerisation) through the incorporation of nucleotide bases in a growing DNA chain (for instance the nascent strand referred to herein), such as primer extension.
• ISFETs are known in the art.
• the ISFET used herein is provided with an ion sensitive layer such as silicon nitride, on top of which a layer of polymerase may be provided.
• the magnitude of pH change is detected in order to reliably detect nucleotide insertion (elongation of the nascent polynucleotide strand).
• the change in electrical signal of the ISFET is indicative of nucleotide insertion during DNA synthesis.
• a pyrophosphate (PPi or P 2 0 7 4+ ) ion is released.
• Nucleic acid polymerisation can be found in many biological reactions such as cell replication, gene expression, or DNA repair. The reaction generally occurs with the help of enzymes (e.g. polymerase). Technologies have been developed to replicate the nucleic acid replication reaction outside host cells or organisms. This has many applications such as amplification of nucleic acid in vitro, sequencing of nucleic acid, genotyping, and molecular diagnosis.
• a typical nucleic acid polymerisation can involve triphosphate deoxynucleotides and a DNA substrate as expressed below.
• x molecules of dNTPs are hydrolysed into x molecules of pyrophosphate and the DNA is extended from y nucleotides to x+y nucleotides.
• the reaction can also involve nucleotides and RNA and the nucleic acid substrate can be double stranded or single stranded. The actual number of hydrogen ions released depending on various factors such as pK values of the reagents.
• This polymerisation reaction occurs when the 5' end of a nucleotide is incorporated onto the 3' (extending) end of a polynucleic acid annealed to a second (template or target) polynucleic acid, the sequence or nucleic acid identity of which is to be determined.
• the incorporation will only occur if the nucleotide is complementary to the base of the second polynucleic acid (i.e. the template strand) opposite the point of incorporation (i.e. Thymine/Uracil is complementary to Adenine and Guanine is complementary to Cytosine).
• the primary reaction is dependent on the DNA successfully polymerising, which occurs if the nucleotides or primer added are complementary to the unknown template strand of the DNA to be sequenced (i.e. the bases of one or each of nucleotide are identified, for example as being C, G, A, T, or U).
• the secondary reactions depend on byproducts being produced by the primary reaction. Therefore, the overall reaction produces (i.e. results in a net release of) protons if the nucleotides or primer added are complementary to the unknown template DNA. These protons are detected by the ISFET and correlated to the addition of the specific nucleotides and primer to identify the original DNA in the sample. Thus, knowing which nucleotide or primer was added to extend the nascent strand will indentify a portion of the unknown (template) nucleic acid.
• the polymerisation reaction may result from hybridising the unknown nucleic acid with an allele specific primer and mixed dNTPs.
• a primer may be used to anneal to the unknown nucleic acid up to a location of interest followed by adding a known dNTP.
• the polymerisation reaction may be an allele- specific amplification reaction using PCR or isothermal amplification.
• the use of an allele-specific primer allows for identifications such as SNPs, i.e. the determination of the presence or absence of a particular SNP of the identity of the nucleotide at the relevant position, to thereby identity an allele in a sample.
• the hydrolysis of dNTP to PPi causes changes in proton concentration within certain pH ranges and with the presence of divalent metal ions, such as magnesium or manganese.
• inorganic PPase may be used to catalyze the hydrolysis of inorganic PPi to form orthophosphate, as expressed below:
• Pyrophosphate is generally stable for a day or longer without the help of enzymes.
• Preferred reactions are polymerase chain reaction, sequencing, or primer extension. In such reactions, the pyrophosphate remains stable in the reaction mixture.
• pH change is a function of the reaction and reaction percentage. By adding an enzyme or enzymes that hydrolyse pyrophosphate, but not triphosphate acid, a greater pH change can be added (on top of that from the hydrolysis of dNTP).
• Pyrophosphatases are acid anhydride hydrolases that act upon diphosphate bonds. Enzymes such as inorganic pyrophosphatase or thermostable inorganic pyrophosphatase fulfill such criteria. Pyrophosphatases are widely available and may be mixed with polymerase in the polymerisation reaction. In the case of sequencing, the pyrophosphatase is used in tandem with a DNA polymerase to increase the pH change.
• thermostable pyrophosphatase is mixed with thermostable DNA polymerase in a PCR reaction.
• the pyrophosphatase catalyses the hydrolysis of PPi, which is the product of polymerisation.
• the hydrolysis of the pyrophosphate previously considered a waste product at best, thus increases the total pH change (i.e. the concentration of hydrogen ions) from a single nucleotide addition.
• a method of PCR amplification comprising use of PPase to augment or enhance signal detection from an ISFET upon nucleoside addition is also provided.
• the effect of the reaction is demonstrated in the Figure 2.
• the change in pH is measured for 350 seconds for 4 reactions.
• Line 4 shows a reaction having neither Pyrophosphate nor pyrophosphatase; the pH changes very little.
• Line 2 shows a reaction having no Pyrophosphate but some pyrophosphatase; the pH changes initially in reaction to the added pyrophosphatase buffer but then decreases until equilibrium, the offset merely indicative of the buffer addition and not proton release from a reaction.
• Line 1 shows a reaction having both Pyrophosphate and pyrophosphatase; the change in pH increases until equilibrium.
• the reagents used in the reaction comprise KCI, MgCI2, PPiase. Preferred concentrations are provided below.
• the concentration of KCI is greater than 10mM, more preferably greater than 40mM, 50mM, 80mM, 100mM or 120mM.
• concentration is less than 500mM, more preferably less than 400mM, 300mM, or 200mM. Any combination of these upper and lower limits of these is envisaged.
• the concentration of MgCI2 is greater than 1 mM, more preferably greater than 2mM, 3mM, or 4mM. Preferably said concentration is less than 10mM, more preferably less than 8mM, 7mM, or 5mM. Any combination of these upper and lower limits is envisaged.
• the amount of PPiase in a 50uL reaction volume is greater than 0.01 U (where U is defined by an unit of enzymatic activity under conditions prescribed by the manufacturer), more preferably greater than .05U, 0.1 U, 1 U or 10U.
• Suggested pyrophosphatase compounds include Inorganic PPiase made from E. coli or yeast and/or Thermostable Inorganic PPiase both available from New England Biolab (Catalogue no.M0361 S is preferable). Excessive commercially available PPiase could lead to buffering which is undesirable in the detection of hydrogen ions.
• PPiase is provided without any buffer components.
• a typical reaction may occur in a micro-chamber having a volume from 1 nl_ to 100uL, but the volume may be bigger depending on the size of the ISFET and sample volume available.
• the temperature of the reaction volume may be 18-45°C, preferably greater than 25 q C, 30°C, or 35 ⁇ , preferably less than 40°C or 38°C. Any combination of these upper and lower limits is envisaged.
• the pH of the fluid including the DNA sample and reagents after nucleotide incorporation is preferably between 7 and 8.6; more preferably above pH 7.5 or above pH7.9; preferably below pH 8.4 or pH 8.1 . Any combination of these upper and lower limits is envisaged. NaOH may be added to the fluid as required to achieve the above pH setting. Other appropriate buffers are also contemplated.
• the by-product DNA may also be deconstructed to release protons.
• the reaction may be expressed as:
• dNMP is a deoxymonophosphate and the DNA is a newly synthesised (nascent) polynucleic acid complementary to the unknown DNA template.
• the present enzyme may be a DNA deconstruction enzyme preferably a hydrolysis enzyme such as an exonuclease, preferably being capable of using DNA and/or RNA as its substrate. Any reference to DNA herein also applies to RNA and other polynucleotides unless otherwise apparent.
• the DNA deconstruction enzyme i.e. an exonuclease
• the DNA deconstruction enzyme enzyme is added after the production of identifiable DNA.
• protons released from the deconstruction reaction represent those derived from deconstruction (preferably hydrolysis) of the identifiable DNA and not other DNA fragments in the reaction mixture.
• DNA is identifiable when produced from an allele- specific polymerisation reaction. If the unknown DNA does not polymerise there will still be some deconstruction activity so it is preferable to amplify the quantity of identifiable DNA. This may be done using allele specific amplification techniques such that the quantity of DNA that can be identified is orders of magnitude greater than the unamplified DNA.
• the reagents used in the reaction comprise KCI, MgCI2, Bovine serum albumin (BSA), and DNA deconstruction enzyme. Preferred concentrations are provided below.
• the concentration of KCI is greater than 10mM, more preferably greater than 10mM, 20mM, 30mM, 40mM or 50mM.
• concentration is less than 500mM, more preferably less than 400mM, 300mM, or 200mM. Any combination of these upper and lower limits is envisaged.
• the concentration of MgCI2 is greater than 1 mM, more preferably greater than 2mM, 3mM, or 4mM. Preferably said concentration is less than 10mM, more preferably less than 8mM, 7mM, or 5mM. Any combination of these upper and lower limits is envisaged.
• a typical reaction may occur in a micro-chamber having a volume from 1 nl_ to 100uL, but the volume may be bigger depending on the size of the ISFET and sample volume available.
• the temperature of the reaction volume may be 18-50°C, preferably greater than 25 °C, 30°C, or 35 ⁇ , preferably less than 40°C or 38°C. Any combination of these upper and lower limits is envisaged.
• the pH of the fluid including the DNA sample and reagents after nucleotide incorporation is preferably between 7 and 9; more preferably above pH 7.5, pH 8, or pH 8.3; preferably below pH 9, pH 8.6 or pH 8.5.NaOH may be added to the fluid as required to achieve the above pH setting. Any combination of these upper and lower limits is envisaged.
• the concentration of BSA is between 0.1 and 10 mg/ml, preferably greater than 0.2 mg/ml, 0.5 mg/ml, or 1 .0 mg/ml; preferably less than 5 mg/ml, 2.0 mg/ml or 1 .5 mg/ml. Any combination of these upper and lower limits is envisaged.
• the enzyme used may be any enzyme that deconstructs DNA, preferably by the hydrolysis of phosphate/phosphodiester bonds, and releases protons.
• exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain via a hydrolyzing reaction that cleaves phosphodiester bonds at either the 3' or the 5' end.
• the enzyme is Exonucelase III or Lambda exonuclease, both available from New England Biolab or Fermentas.
• Preferred examples are Exonuclease III from Fermentas (EN0191 ) and Lambda Exonuclease from Fermentas (EN0562).
• the quantity of Exonuclease per 50ul is greater than 10U, 20U, 50U, or 100U.
• the quantity of Lambda Exonuclease per 50ul is greater than 1 U, 2U, 5U, or 10U.
• Figure 4 is a graph of the pH of a reaction chamber with and without Exonuclease III.
• 10 ng/uL of dsDNA substrate was mixed with Exonuclease III to produce protons with the shown effect on pH (shown by the digested DNA - solid line).
• the control experiment (shown by the non-digested DNA - dotted line), contains all reagents except the enzyme.
• the pH will initially drop due to the addition of reagents to the reaction chamber thereafter becoming relatively unchanged for the control or dropping further for the enzyme reaction.
• Figure 3 shows that lambda Exonuclease has a similar effect.
• the amount of DNA present in the sample to be tested may vary or be unknown but is typically between 50 and150 ng/uL preferably more than 10 ng/uL, more than 20 ng/uL or more than 50 ng/uL.
• Nucleic acid deconstruction may be used to enhance the pH signal from samples comprising purified nucleic acid, or nucleic acid from a previous enzymic reactions. Therefore the method can tolerate background compounds, such as leftover primers/probes (up to 1 microM), oligo nucleotide (up to 10 mM), DNA polymerases, and other reaction components or by-products.
• the invention has been described in terms of preferred embodiments as set forth above, it should be understood that these embodiments are illustrative only and that the claims are not limited to those embodiments. Those skilled in the art will be able to make modifications and alternatives in view of the disclosure which are contemplated as falling within the scope of the appended claims.
• Each feature disclosed or illustrated in the present specification may be incorporated in the invention, whether alone or in any appropriate combination with any other feature disclosed or illustrated herein.
• the enzymes to deconstruct by-products may comprise both pyrophosphatase and an enzyme to deconstruct DNA, to release protons from the primary polymerisation reaction and both deconstruction reactions, such that the signal change is even larger.
• nucleotide and nucleic acid are used interchangeably herein. Either is preferred.
• the present invention therefore provides a means of enhancing an ionic signal, i.e. a change in pH caused by release of hydrogen ions, such release being indicative of an addition or the removal of a nuclei acid from a nucleic acid strand.
• an ionic signal i.e. a change in pH caused by release of hydrogen ions, such release being indicative of an addition or the removal of a nuclei acid from a nucleic acid strand.

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